The Ribo-Zero kits were developed using model organisms. Results can vary depending on the species from which the RNA sample was derived, especially with 5S rRNA removal.
Use the RNAMatchMaker tool to confirm species compatibility.
See our species compatibility tables. If you do not see your species in the list please use RNAMatchMaker to learn if a current Ribo-Zero kit will match your organism.
No. Our sequences are proprietary and we are unable to share them.
The Ribo-Zero Gold Kit contains additional capture probes that cover mitochondrial rRNA sequences, which account for about 7% of the rRNA in most mammalian cells. The standard Ribo-Zero Kit will remove cytoplasmic (nuclear-encoded) rRNA, but not mitochondrial rRNA.
Yes. While there is a possibility of some cross-hybridization between the capture probes and minute portions of other RNA species, the hybridization conditions are optimized for rRNA capture while minimizing undesirable cross-hybridization.
Poly(A) enrichment involves the capture of the 3´-end of polyadenylated RNA, typically using oligo(dT) columns. If the RNA to be enriched is of high quality (RIN >9), then most of the mRNA will be recovered. However, if the input RNA is of poor quality (RIN <6), then the majority of the mRNA sequences will be lost and the resulting mRNA-Seq libraries will contain only 3´ sequences of the mRNA. By using the Ribo-Zero procedure, capture of the rRNA (intact or degraded) does not affect the mRNA sequences in the original RNA preparation. Libraries prepared after Ribo-Zero treatment therefore exhibit more even coverage density, and greatly reduced 3´ end bias compared to poly(A) enrichment. R-Z treatment also recovers non-poly(A), non-rRNA such as lincRNA that Poly(A) enrichment loses.
Yes. The Ribo-Zero Bacteria Kit contains capture probes for both Gram-positive and Gram-negative bacteria, thus allowing removal of rRNA from an RNA sample that contains transcripts from many bacterial species.
No. We retained the non-magnetic (filter-based) “Core Kits” until December 2015 and are currently no longer available.
Ribo-Zero kits remove rRNA from partially degraded samples. However, probes cannot hybridize to severely degraded samples and are therefore not effective.
RNA samples must be treated with DNase before starting the rRNA removal protocol. Otherwise, the probes can bind to the DNA and reduce rRNA removal.
You can use as low as 100 ng in the standard Ribo-Zero Kits. Just follow the instructions for 1-2.5 µg input listed in the reference guide.
You can purchase a kit for each of the organisms and then mix the rRNA removal solution in an equal ratio. Then proceed with the protocol as stated in the user guide. You may want to decrease your input amount to 2.5 ug to ensure the rRNA is removed fully.
The Ribo-Zero Kits work very well on FFPE RNA. The design and configuration of the probes allows for the capture of very small pieces of rRNA, leaving the mRNA and other non-ribosomal species in the supernatant.
Ribo-Zero kits remove ribosomal RNA (rRNA) using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The probe:rRNA hybrid is then captured by magnetic beads and removed using a magnet, leaving the desired rRNA-depleted RNA in solution.
Follow the protocol as described in the reference guide for your kit. The procedures require frequent and complete mixing for successful rRNA removal.
The appropriate volume of removal solution depends on the amount of total RNA input. Accurately quantify the amount of RNA and see the kit refence guide for instructions on determining the correct volume of solution.
The magnetic beads must be at room temperature before they are used in wash steps. After the wash, keep beads at room temperature for use in rRNA removal.
If frozen, the magnetic beads do not work. Confirm that the beads were not frozen when they arrived in the lab, and that they were properly stored at 2°C to 8°C.
Make sure that you add the RNA sample to the washed, room-temperature magnetic beads. Do not add magnetic beads to the RNA sample.
Magnetic beads can clump after the probe-hybridized RNA sample is added. This happens because the probes contain biotin that can bind to multiple magnetic bead particles. Make sure that you vortex the beads vigorously for at least 10 seconds.
Failure to remove the magnetic beads completely causes carryover of the probes and rRNA. Place the tubes on a magnetic stand for 5 minutes, and then carefully transfer the supernatant containing the rRNA-depleted RNA to a new RNase-free tube.
No, the beads will no longer work. You must purchase a replacement kit.
No, please speed vac your sample to the appropriate volume but do not dry the sample down completely.
The RNA needs to be purified for Ribo-Zero to work properly. If you use RNA that is not clean the salts will change the conditions of the reaction in the Ribo-Zero kit.
Figure 1. A. S. cereviseae samples from 2 mm colonies on solid YPD media was purified using the MasterPure™ Yeast RNA Purification Kit with DNase treatment, to achieve final yield of 3 µg RNA. B. 1 µg of purified RNA was processed using the Ribo-Zero™ Magnetic Gold Kit (Yeast) to remove the 18S and 25S rRNA. Results are similar with other organisms
This is normal and expected. The peaks observed in the 100- to 140-nt region on a Bioanalyzer trace correspond to tRNA, small noncoding RNAs, and small amounts of 5S rRNA (in cases where 5S rRNA removal is less efficient). These small RNAs are available for inclusion in a sequencing library if desired, but can be readily removed by gel or other size-selection techniques that are designed to remove small RNAs, either before or after library synthesis.